1. Field of the Invention
The present invention relates to treatment of cancer in mammals. Generally, the present invention provides methods of treating cancer in a mammal by administering a polynucleotide construct comprising a polynucleotide encoding a cytokine. In addition, the present invention relates to the methodology for selective transfection of malignant cells with polynucleotides expressing therapeutic or prophylactic molecules in intra-cavity tumor bearing mammals. More specifically, the present invention provides a methodology for the suppression of an intra-cavity dissemination of malignant cells, such as intraperitoneal dissemination.
The present invention further relates generally to compositions and methods useful for in vivo polynucleotide-based polypeptide delivery into cells of vertebrates. More particularly, the present invention provides the use of sodium phosphate solutions in compositions and methods useful for direct polynucleotide-based polypeptide delivery into the cells of vertebrates.
2. Related Art
Cytokines have been demonstrated both in pre-clinical animal models as well as in humans to have potent anti-tumor effects. In particular IFN's have been tried for the treatment of a number of human concerns.
The interferons (IFNs) are a family of cytokines with potent anti-viral, antiproliferative, and immunomodulatory activities and play important roles in the body's defensive response to viruses, bacteria, and tumors (Baron, S. et al., JAMA 266:1375 (1991)). On the basis of antigenicity, biochemical properties, and producer cell, the interferon's have been divided into two classes, type I interferon and type II interferon. IFNα, IFNβ, IFNω, and IFNτ are type I interferons, and bind to the same α/β receptor. IFNγ is a type II interferon, and binds to the γ receptor (Pestka, S., Ann. Rev. Biochem. 56:727 (1987)). IFNα and IFNβ are naturally expressed in many cells upon viral infection. IFNγ is produced by activated T lymphocytes and natural killer (NK) cells. IFNτ is believed to possess hormone activity, and plays an important role in pregnancy in cattle, sheep, and related ruminants (Imakawa, K. et al., Nature 330:377 (1987); Stewart, H. J. et al., J. Endocrinology 115:R13 (1987)). Due to the pleiotropic activities of IFNs, these cytokines have been studied for their therapeutic efficacy in a number of diseases, particularly cancers and viral infectious diseases.
IFNω was discovered independently by three different groups in 1985 (Capon, D. J., et al., Molec. Cell. Biol. 5: 768-779 (1985), Feinstein, S. et al., Molec. Cell. Biol 5:510 (1985); and Hauptmann and Swetly, Nucl. Acids Res. 13: 4739-4749 (1985)). Unlike IFNα, for which at least 14 different functional nonallelic genes have been identified in man, IFNω is encoded by a single functional gene. IFNω genes are believed to be present in most mammals, but have not been found in dogs, rats or mice. The mature IFNω polypeptide is 172 amino acids and shares 60% sequence homology with the human IFNα's. Due to the sequence similarity with IFNα, IFNω was originally considered to be a member or a subfamily of IFNα, and was originally termed IFNα-II. IFNω is a significant component (≈10%) of human leukocyte-derived interferon, the natural mixture of interferon produced after viral infection (Adolf, G. et al., Virology 175:410 (1990)). IFNω has been demonstrated to bind to the same α/β receptor as IFNα (Flores, I. et al., J. Biol. Chem. 266: 19875-19877 (1991)), and to share similar biological activities with IFNα, including anti-proliferative activity against tumor cells in vitro (Kubes, M. et al., J. Interferon Research 14:57 (1994) and immunomodulatory activity (Nieroda et al., Molec. Cell. Differentiation 4: 335-351 (1996)).
Recombinant IFNα polypeptide has been approved for use in humans for hairy cell leukemia, AIDS-related Kaposi's sarcoma, malignant melanoma, chronic hepatitis B and C, chronic myleogenous leukemia, and condylomata acuminata (Baron, S. et al., JAMA 266:1375 (1991)). However, for each of these indications, IFNα polypeptide must be administered repeatedly, often on a daily basis, for extended periods of time to maintain effective serum levels due to the short half-life (hours) of the polypeptide in the serum (Friedman, Interferons: A Primer, Academic Press, New York, pp. 104-107 (1981); Galvani and Cawley, Cytokine Therapy, Cambridge University Press, Cambridge, pp. 114-115 (1992)). Thus, in spite of producing clinical benefit for many disease conditions, the use of IFNα polypeptide is associated with acute and chronic side effects in most patients (Jones, Cancer 57: 1709-1715 (1986); and Quesda et al., Blood 68: 493-497 (1986)). The severity of the adverse reaction correlates with peak serum interferon levels.
Viral or plasmid vectors containing IFNα genes have been used in ex vivo therapy to treat mouse tumors. For example, tumor cells were transfected in vitro with viral or plasmid vectors containing an IFNα gene, and the transfected tumor cells were injected into mice (Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994); Kaido, T. et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H. et al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997)). In another ex vivo study, cervical carcinoma and leukemia cells were transfected with a viral vector containing the interferon-consensus gene, and the transfected cells were injected into mice (Zhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 (1996)). In all of these ex vivo studies, varying levels of anti-tumor efficacy, such as tumor regression and/or prolonged survival, have been observed.
Viral or plasmid vectors containing interferon genes have also been used in in vivo therapy for tumor-bearing mice. For example, a viral vector containing the interferon-consensus gene was injected into mice bearing transplanted MDA-MB-435 breast cancer, hamster melanoma, or K562 leukemia, and tumor regression was reported (Zhang, J.-F. et al., Proc. Natl. Acad. Sci. USA 93: 4513-4518 (1996)). In a similar study, a plasmid vector containing human IFNβ gene complexed with cationic lipid was injected intracranially into mice bearing a human glioma, and tumor regression was reported (Yagi, K. et al., Biochemistry and Molecular Biology International 32: 167-171 (1994)). In a murine model of renal cell carcinoma the direct intratumoral injection of an IL-2 plasmid DNA:lipid complex has been shown to result in complete tumor regression and a significant induction of a tumor specific CTL response increase in survival (Saffran et al., Cancer Gene Therapy 5: 321-330 (1998)).
Plasmid vectors containing cytokine genes have also been reported to result in systemic levels of the encoded cytokine and in some cases, biological effects characteristic of each cytokine in mice. For example, the intramuscular injection of plasmid DNA encoding either TGFβ, IL-2, IL-4, IL-5, or IFNα resulted in physiologically significant amounts in the systemic circulation of the corresponding cytokine polypeptide (Raz, E. et al., Proc. Natl. Acad. Sci. USA 90: 4523-4527 (1993); Raz, E. et al., Lupus 4: 266-292 (1995); Tokui, M. et al., Biochem. Biophys. Res. Comm. 233: 527-531 (1997); Lawson, C. et al., J. Interferon Cytokine Res. 17: 255-261 (1997); Yeow, W.-S. et al., J. Immunol. 160: 2932-2939 (1998)).
U.S. Pat. No. 5,676,954 reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355; 4,946,787; 5,049,386; 5,459,127; 5,589,466; 5,693,622; 5,580,859; 5,703,055; and International Patent Application No. PCT/US94/06069 (publication no. WO 94/29469) provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international patent application no. PCT/US94/06069 (publication no. WO 04/9469) provide methods for delivering DNA-cationic lipid complexes to mammals.
Even though some viral vectors used in ex vivo and in vivo cancer therapy in murine models showed anti-tumor efficacy, the use of viral vectors to deliver interferon-expressing genes in vivo could induce anti-viral immune responses or result in viral integration into host chromosomes, causing disruption of essential host genes or activation of oncogenes (Ross et al., Human Gene Therapy 7: 1781-1790 (1996)).
For treatment of multiple metastatic carcinomas of a body cavity are treated using laparoscopy (Childers et al, Gynecol. Oncol. 59: 25-33, (1995)), catheterization (Naumann et al, Gynecol. Oncol. 50: 291-3, (1993)) or other access devices (Almadrones et al, Semin. Oncol. Nurs. 11: 194-202, (1995)). Treatment is usually by surgical removal of primary and large metastatic tumors and postoperative chemotherapy (Kigwawa et al, Am. J. Clin. Oncol. 17: 230-3, (1994); Markman et al, J. Clin. Oncol. 10: 1485-91, (1992)) or radiotherapy (Fjeld et al, Acta. Obstet. Gynecol. Scand Suppl. 155: 105-11, (1992)). Tumor recurrence is monitored by magnetic resonance imaging (Forstner et al, Radiology 196: 715-20, (1995)), ascites cytology (Clement, Am. J. Clin. Pathol. 103: 673-6, (1995); Forstner et al, Radiology 196: 715-20, 1995) and blood analyses (Forstner et al, Radiology 196: 715-20, (1995)). Many intraperitoneal (i.p.) cancers, such as ovarian cancer, eventually metastasize via the lymphatic system to the lungs or other vital organs, and the prognosis for the patient is very poor (Kataoka et al, Nippon Sanka Fujinka Gakkai Zasshi 46: 337-44, 1994; Hamilton, Curr. Probl. Cancer 16: 1-57, (1992)).
Human ovarian cancer is often diagnosed at an advanced stage when the effectiveness of surgery and chemotherapy are limited. The lack of effective treatment options for late-stage patients warrants the development of new treatment modalities for this disease. There have been several attempts to develop an effective immunotherapy for the treatment of ovarian cancer.
The early work in this area involved mouse studies in which bacteria-derived immunostimulants, such as Bacillus Calmette-Guerin (BCG) and Corynebacterium parvum, were injected i.p. as non-specific activators of the immune system. (Knapp and Berkowitz, Am. J. Obstet. Gynecol., 128: 782-786, (1977); Bast et al., J. Immunol., 123: 1945-1951, (1979); Vanhaelen, et al., Cancer Research, 41: 980-983, (1981); and Berek, et al., Cancer Research, 44, 1871-1875, (1984)). These studies generally resulted in a non-specific immune response that often did not prevent the growth of later tumors. In addition, if the bacterial antigens were injected more than 24 hours after tumor cell inoculation, there was minimal antitumor response, suggesting that treatment of late-stage ovarian cancer patients with this type of therapy would not be effective.
More recent studies in both mice and humans have involved the i.p. or intravenous (i.v.) administration of cytokine proteins as more specific activators of the immune response (Adachi, et al, Cancer Immunol. Immunother. 37: 1-6, (1993); Lissoni, et al, Tumori. 78: 118-20, (1992)). Treating murine ovarian tumors with a combination of recombinant IL-2 and GM-CSF proteins had some beneficial effect in inhibiting ascites production; however, IL-2 was only effective if it was combined with GM-CSF (Kikuchi, et al., Cancer Immunol. Immunother., 43: 257-261, (1996)). Similarly, a combination of IL-2 and lymphokine-activated killer (LAK) cells was able to reduce i.p. sarcomas in mice, while IL-2 protein alone was not as effective (Ottow, et al., Cellular Immunology, 104: 366-376, (1987)). Human clinical trials evaluating IL-2 protein therapy of ovarian cancer patients indicated some antitumor effects (Chapman et al., Investigational New Drugs, 6:179-188, (1988); West et al., N. Engl. J. Med. 316:898-905, 1987; Lotze et al., Arch. Surg. 121:1373-1379, 1986; Benedetti Panici et al., Cancer Treatment Review, 16A:123-127, 1989; Beller et al., Gynecol. Oncol., 34:407-412, 1989; Urba et al., J. Natl. Cancer Inst., 81:602-611, 1989; Stewart et al., Cancer Res., 50:6302-6310, 1990; Steis et al., J. Clin. Oncol., 8:1618-1629, 1990; Lissoni et al., Tumori, 78:118-120, 1992; Sparano et al., J. of Immunotherapy, 16:216-223, 1994; Freedman et al., J. of Immunotherapy, 16:198-210, 1994; Edwards et al., J. Clin. Oncol., 15:3399-3407, 1997).
Recent studies in mice have involved the injection of DNA constructs encoding “suicide” genes followed by treatment with prodrugs. This approach has successfully caused regression of some small tumors but has been less successful on larger tumor masses. (Szala, et al. Gene Therapy 3: 1025-1031, 1996; Sugaya, et al. Hum Gene Ther 7: 223-230 (1996)). In another study, liposome-mediated E1A gene therapy for mice bearing ovarian cancers that overexpress HER-2/neu resulted in reduced mortality among these tumor bearing mice. (Yu, et al. Oncogene, 11: 1383-1388 (1995)). Similarly, the successful treatment of murine ovarian carcinoma (MOT) has been demonstrated using cisplatin-induced gene transfer of DNA constructs encoding IFNγ via i.p. injection. (Son, Cancer Gene Therapy 4: 391-396 (1997)). However, this study demonstrated that tumors were poorly responsive to either the IFNγ gene or cisplatin alone, suggesting that the effectiveness of the cisplatin-based gene therapy protocol was mainly due to enhanced sensitization of cisplatin-exposed tumor cells to transfection by the IFNγ gene. (Son, Cancer Gene Therapy 4: 391-396, 1997).
Clearly, there is a need for superior therapeutic compositions and methods for treating mammalian cancer. Further, there is a need for an in vivo delivery system for IFNω. The present invention provides a simple and safe yet effective compositions and methods for treatment of mammalian cancer.
The present invention also solves the problems inherent in prior attempts to treat body cavity malignancies. The inventors show herein that the malignant cell dissemination into body cavities, such as into the peritoneal cavity during late stage ovarian cancer, can be suppressed simply by administering as few as two to six doses of a polynucleotide formulation directly into the body cavity. This treatment results in selective transfection of malignant cells, and subsequent long-term local production of an effective amount of therapeutic molecules.
The in vivo delivery of a polynucleotide (e.g., plasmid DNA) into vertebrate tissues has been shown to result in the cellular uptake and expression of the polynucleotide into a desired polypeptide (Wolff, J. A. et al., Science 247:1465-1468 (1990); Wheeler, C. J. et al., Proc. Natl. Acad. Sci. USA 93:11454-11459 (1996)). Potential human therapeutic uses of such polynucleotide-based polypeptide delivery include immune response induction and modulation, therapeutic polypeptide delivery, and amelioration of genetic defects. For example, a polynucleotide may encode an antigen that induces an immune response against an infectious pathogen or against tumor cells (Restifo, N. P. et al., Folia Biol. 40:74-88 (1994); Ulmer, J. B. et al., Ann. NY Acad. Sci. 772:117-125 (1995); Horton, H. M. et al., Proc. Natl. Acad. Sci. USA 96:1553-1558 (1999); Yagi, K. et al., Hum. Gene Ther. 10: 1975-1982 (1999)). The polynucleotide may encode an immunomodulatory polypeptide, e.g., a cytokine, that diminishes an immune response against self antigens or modifies the immune response to foreign antigens, allergens, or transplanted tissues (Qin, L. et al., Ann. Surg. 220:508-518 (1994); Dalesandro, J. et al., J. Thorac. Cardiovasc. Surg. 111: 416-421 (1996); Moffatt, M. and Cookson, W., Nat. Med. 2:515-516 (1996); Ragno, S. et al., Arth. and Rheum. 40:277-283 (1997); Dow, S. W. et al., Hum. Gene Ther. 10:1905-1914 (1999); Piccirillo, C. A. et al., J. Immunol. 161:3950-3956 (1998); Piccirillo, C. A. and Prud'homme, G. J., Hum. Gene Ther. 10: 915-1922 (1999)). For therapeutic polypeptide delivery, the polynucleotide may encode, for example, an angiogenic protein, hormone, growth factor, or enzyme (Levy, M. Y. et al., Gene Ther. 3:201-211 (1996); Tripathy, S. K. et al., Proc. Natl. Acad. Sci. USA 93:10876-10880 (1996); Tsurumi, Y. et al., Circulation 94:3281-3290 (1996); Novo, F. J. et al., Gene Ther. 4:488-492 (1997); Baumgartner, I. et al., Circulation 97:1114-1123 (1998); Mir, L. M. et al., Proc. Natl. Acad. Sci. USA 96:4262-4267 (1999)). For amelioration of genetic defects, the polynucleotide may encode normal copies of defective proteins such as dystrophin or cystic fibrosis transmembrane conductance regulator (Danko, I. et al., Hum. Mol. Genet. 2:2055-2061 (1993); Cheng, S. H. and Scheule, R. K., Adv. Drug Deliv. Rev. 30:173-184 (1998)).
However, the efficiency of a polynucleotide uptake and expression, especially when the polynucleotide is not associated with infectious agents, is relatively low. For example, Doh, S. G. et al., Gene Ther. 4:648-663 (1997) reports that the administration of plasmid DNA into mouse muscle results in the detectable transduction of an average of only 6%, e.g., about 234 out of approximately 4000, of the myofibers in the injected muscle. Wheeler, C. G. et al., ibid., showed that administration of plasmid DNA complexed with cationic lipid into a mouse lung results in the transduction of less than 1% of the lung cells.
Attempts have been made to increase the efficiency of in vivo polynucleotide administration into vertebrates using chemical agents or physical manipulations. Such chemical agents include cellular toxins such as bupivacaine or barium chloride (Wells, D. J., FEBS Letters 332:179-182 (1993); Vitadello, M. et al., Hum. Gene. Ther. 5:11-18 (1994); Danko, I. et al., Hum. Mol. Genet. 2:2055-2061 (1993)) which act to cause muscle damage followed by muscle regeneration by cell division which makes the cells more receptive to DNA entry (Thomason, D. B. and Booth, F. W., Am. J. Physiol. 258:C578-81 (1990)); polymers such as polyvinyl pyrollidine that coat the DNA and protect it from DNases (Mumper, R. J., et al., Pharm. Res. 13:701-709 (1996); Mumper R. J. et al., J. Cont. Rel. 52:191-203 (1998); Anwer, K. et al., Pharm. Res. 16:889-95 (1999)); bulking agents such as sucrose that are injected before DNA injection to help expand the spaces between muscle cells and therefore allow better distribution of the subsequently injected DNA (Davis, H. L. et al., Hum. Gene Ther. 4:151-159 (1993)); DNA binding agents such as histones or intercalaters that protect the DNA from DNases (Manthorpe, M. et al., Hum. Gene Ther. 4:419-431 (1993); Wolff, J. A., Neuromuscul. Disord. 7:314-318 (1997); WO 99/31262). Physical manipulations include removal of nerves that control muscle contraction (Wolff, J. A. et al., BioTechniques 11:575-485 (1991)); electroporation that electrically opens muscle cell pores allowing more DNA entry (Aihara, H. and Miyazaki, J., Nature Biotechnol. 16:867-870 (1998); Mir, L. M. et al., CR Acad Sci. III 321:893-899 (1998), Mir, L. M. et al., Proc. Natl. Acad. Sci. USA 96:4262-4267 (1999); Mathiesen, I., Gene Ther. 6:508-514 (1999); Rizzuto, G. et al., Proc. Natl. Acad. Sci. USA 96:6417-6422 (1999)); use of intravascular pressure (Budker, V. et al., Gene Ther. 5:272-276 (1998)); use of sutures coated with plasmid DNA (Labhasetwar, V. et al., J. Pharm. Sci. 87:1347-1350 (1998); Qin, Y. et al., Life Sci. 65:2193-2203 (1999)); use of sponges soaked with DNA as intramuscular depots to prolong DNA delivery (Wolff, J. A. et al. (1991), ibid.); use of special needle-based injection methods (Levy, M. Y. et al., Gene Ther. 3:201-211 (1996); Doh, S. G. et al. (1997), ibid.); and of needleless-injectors that propel the DNA into cells (Gramzinski, R. A. et al., Molec. Med. 4:109-118 (1998); Smith, B. F. et al., Gene Ther. 5:865-868 (1998); Anwer, K. et al. (1999) ibid.). In addition, Wolff, J. A. et al. (1991) ibid. and Manthorpe, M. et al. (1993) ibid. refers to conditions affecting direct gene transfer into rodent muscle in vivo.
WO99/64615 identifies the use of products and methods useful for delivering formulated nucleic acid molecules using electrical pulse voltage delivery. Examples include the formulation of plasmid DNA in a saline solution containing agents that promote better delivery of the plasmid DNA into cells in vivo when the formulation is delivered with an electrical pulse. Electrical pulse delivery often comprises electroporation where an electrical pulse is delivered to a tissue that is previously injected with a drug. Electroporation of a tissue causes transient interruption of cell membranes allowing more drug to enter the cell through the interruptions or “pores”. The agents in the saline DNA solution that promote delivery of the DNA into electroporated tissues include propylene glycols, polyethylene glycols, poloxamers (block copolymers of propylene oxide and ethylene oxide), or cationic lipids. They claim that the way that these agents enhance delivery of the DNA into cells is by either protecting the DNA from degradation by DNases or by condensing the DNA into a smaller form, or both.
Many of these attempts to enhance tissue transduction have used agents that destroy muscle (bupivacaine, barium chloride) and actually lower expression (Norman, J. et al., Methods in Molec. Med. 29:185-196 (1999)); have to be pre-injected before the DNA (sucrose); are expensive organic polymers (polyvinyl pyrollidine), mutagens (intercalaters), antigenic proteins (histones) or devices that destroy muscle tissue (needless or needle-free injectors); or need to be inserted surgically (sutures, sponges, intravascular pressure). Furthermore, most of these methods may be expensive and not suitable or practical for human use.
On the other hand, little attention has been given to the use of alternative salt solutions and/or auxiliary agents in the pharmaceutical formulation as a way of enhancing the efficiency of a polynucleotide-based polypeptide delivery. Investigators in this field routinely use normal saline or phosphate buffered saline (PBS 0.9% (i.e., about 154 mM) NaCl and 10 mM Na-phosphate) solutions for polynucleotide delivery, e.g., by intramuscular injection, because they are physiologically isotonic, isoosmotic, stable, non-toxic, and also because they have been traditionally used for human intramuscular injections of other drugs. However, sodium phosphate, in the absence of saline, has been used in humans for delivery of non-polynucleotide-based drugs (e.g., small molecules) administered via the intramuscular or intravenous routes (See generally, Physician's Desk Reference. Medical Economics Co, Monyvale, N.J. (1998)).
Sodium or potassium phosphate have been reported to enhance Lipofectin™-mediated transfection of human osteosarcoma cells in vitro (Kariko, K., et al., Biochim Biophys Acta 1369:320-334 (1998)), and the use of RPMI cell culture medium buffered with NaHCO3/Na2HPO4 were reported to be the best medium for forming DNA/cationic lipid complexes in vitro. (Kichler, A., et al., Gene Ther. 5:855-860 (1998)).
There remains a need in the art for a convenient and safe way of improving the effectiveness of in vivo polypeptide delivery via direct administration of a polynucleotide. Aqueous solutions of certain salts including sodium phosphate have been used in humans (i.e., intramuscular injection of various small molecule drugs), and detergents or surfactants as auxiliary agents are common additives in drugs administered into human tissues. However, the use of certain salts or auxiliary agents, or a combination thereof to improve the transduction, i.e., the entry into cells, and/or expression-enhancing efficiency of polynucleotides delivered in vivo is new.